Ziyuglycoside II attenuated OVX mice bone loss via inflammatory responses and regulation of gut microbiota and SCFAs.

BACKGROUND AND PURPOSE: Osteoporosis (OP) is a frequent clinical problem for the elderly. Traditional Chinese Medicine (TCM) has achieved beneficial results in the treatment of OP. Ziyuglycoside II (ZGS II) is a major active compound of Sanguisorba officinalis L. that has shown anti-inflammation and antioxidation properties, but little information concerning its anti-OP potential is available. Our research aims to investigate the mechanism of ZGS II in ameliorating bone loss by inflammatory responses and regulation of gut microbiota and short chain fatty acids (SCFAs) in ovariectomized (OVX) mice. METHODS: We predicted the mode of ZGS II action on OP through network pharmacology and molecular docking, and an OVX mouse model was employed to validate its anti-OP efficacy. Then we analyzed its impact on bone microstructure, the levels of inflammatory cytokines and pain mediators in serum, inflammation in colon, intestinal barrier, gut microbiota composition and SCFAs in feces. RESULTS: Network pharmacology identified 55 intersecting targets of ZGS II related to OP. Of these, we predicted IGF1 may be the core target, which was successfully docked with ZGS II and showed excellent binding ability. Our in vivo results showed that ZGS II alleviated bone loss in OVX mice, attenuated systemic inflammation, enhanced intestinal barrier, reduced the pain threshold, modulated the abundance of gut microbiota involving norank_f__Muribaculaceae and Dubosiella, and increased the content of acetic acid and propanoic acid in SCFAs. CONCLUSIONS: Our data indicated that ZGS II attenuated bone loss in OVX mice by relieving inflammation and regulating gut microbiota and SCFAs.

Estrogen receptor GPR30 in the anterior cingulate cortex mediates exacerbated neuropathic pain in ovariectomized mice.

Menopausal women experience neuropathic pain 63% more frequently than men do, which may attribute to the estrogen withdrawal. However, the underlying mechanisms remain unclear. Here, the role of estrogen receptors (ERs) in ovariectomized (OVX) female mice following chronic constriction injury (CCI) was investigated. With 17ß-estradiol (E2) supplemented, aggravated mechanical allodynia in OVX mice could be significantly alleviated, particularly after intra-anterior cingulate cortex (ACC) E2 delivery. Pharmacological interventions further demonstrated that the agonist of G-protein-coupled estrogen receptor 30 (GPR30), rather than ERα or ERß in the ACC, exhibited the similar analgesic effect as E2, whereas antagonist of GPR30 exacerbated allodynia. Furthermore, OVX surgery reduced GPR30 expression in the ACC, which could be restored with estrogen supplementation. Selective downregulation of GPR30 in the ACC of naïve female mice induces mechanical allodynia, whereas GPR30 overexpression in the ACC remarkedly alleviated OVX-exacerbated allodynia. Collectively, estrogen withdrawal could downregulate the ACC GPR30 expression, resulting in exacerbated neuropathic pain. Our findings highlight the importance of GPR30 in the ACC in aggravated neuropathic pain during menopause, and offer a potential therapeutic candidate for neuropathic pain management in menopausal women.

Passive exercise is an effective alternative to HRT for restoring OVX induced mitochondrial dysfunction in skeletal muscle.

Background: Postmenopausal women are more prone to develop muscle weakness, which is strongly associated with impairment of mitochondrial function in skeletal muscle. This study aimed to examine the impact of a passive exercise modality, whole-body vibration training (WBVT), on muscle mitochondrial function in ovariectomized (OVX) mice, in comparison with 17ß-estradiol (E2) replacement. Methods: Female C57BL/6J mice were assigned to four groups: sham operation control group (Sham), ovariectomized group (OVX), OVX with E2 supplement group (OVX+E), and OVX with WBVT group (OVX+W). The estrous cycle, body weight, body composition, and muscle strength of the mice were monitored after the operation. Serum E2 level was assessed by enzyme-linked immunosorbent assay (ELISA). The ATP levels were determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was evaluated using high-resolution respirometry (O2K). Expression levels of oxidative phosphorylation (OXPHOS), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) were detected using western blotting. Results: We observed decreased muscle strength and impaired mitochondrial function in the skeletal muscle of OVX mice. The vibration training alleviated these impairments as much as the E2 supplement. In addition, the vibration training was superior to the ovariectomy and the estradiol replacement regarding the protein expression of PGC-1α and TFAM. Conclusion: WBVT improves the OVX-induced decline in muscle strength and impairment of mitochondrial function in the skeletal muscle. This passive exercise strategy may be useful as an alternative to E2 replacement for preventing menopausal muscular weakness. Further studies are needed to understand the effects of WBVT on various physiological systems, and precautions should be taken when implementing it in patient treatment.

Effects of dietary adjustment of n-3:n-6 fatty-acid ratio to 1:2 on anti-inflammatory and insulin-signaling pathways in ovariectomized mice with high fat diet-induced obesity.

Estrogen deficiency increases the secretion of inflammatory mediators and can lead to obesity. Consequently, estrogen deficiency can cause metabolic syndrome, particularly insulin resistance during menopause. Both fish oil and perilla oil contain n-3 fatty acids, which may regulate several inflammatory cytokines. Additionally, adjusting the dietary n-3:n-6 fatty-acid ratio to 1:2 may help treat or prevent chronic diseases. Therefore, we investigated the effect of anti-inflammatory and insulin-signaling pathways, not solely in relation to the (n-3:n-6 fatty-acid ratio at 1:2), but also considering the origin of n-3 fatty acids found in fish oil and perilla oil, in a mouse model of estrogen deficiency induced by ovariectomy and obesity induced by a high-fat diet (HFD). Female C57BL/6J mice were divided into five groups: sham mice on a normal diet; ovariectomized (OVX) mice on a normal diet (OC); OVX mice on a HFD plus lard oil (OL), fish oil (OF), or perilla oil (OP). The dietary n-3:n-6 ratio in the OF and OP groups was adjusted to 1:2. The results showed OF group exhibited significantly lower abdominal adipose tissue weight, fewer liver lipid droplets, and smaller uterine adipocytes, compared with the OL group. Compared with the OL group, the OF and OP groups exhibited higher oral glucose tolerance and lower serum alanine aminotransferase activity, triacylglycerol levels, and total cholesterol levels. Hepatic JAK2, STAT3, and SOCS3 mRNA expression and p-NF-κB p65 and IL-6 levels were significantly lower in the OF and OP groups than in the OL group. Only the OF group exhibited an increase in PI3K and Akt mRNA expression, decrease in GLUT2 mRNA expression, and considerable elevation of p-Akt. Both fish and perilla oil reduced inflammatory signaling markers. However, only fish oil improved insulin signaling (PI3K, Akt, and GLUT2). Our data suggest that fish oil can alleviate insulin signaling through activating the PI3K-Akt-GLUT2 cascade signaling pathway.

D-Pinitol Ameliorated Osteoporosis via Elevating D-chiro-Inositol Level in Ovariectomized Mice.

A natural sugar alcohol, D-pinitol, has been reported to be a potential compound for osteoporosis treatment via inhibiting osteoclastgenesis. However, research on the effects of pinitol on osteoporosis in vivo is still limited. The present study investigated the protective effects of pinitol on ovariectomized mice and attempted to elucidate this mechanism in vivo. Four-week-old female ovariectomized ICR mice were employed as a postmenopausal osteoporosis model and treated with pinitol or estradiol (E2) for 7 wk. Thereafter, serum calcium content, phosphorus content, tartrate-resistant acid phosphatase (TRAcP) and bone-specific alkaline phosphatase activity (BALP) were measured. Bilateral femurs were isolated, and bone marrow protein was collected through centrifuge. Dry femurs were weighed, while femur length, cellular bones, and bone mineral content were measured. D-chiro-Inositol (DCI) and myo-inositol (MI) content in serum and bone marrow was measured by GC-MS. At the end of experiment, the serum BALP and TRAcP activities of the OVX mice were suppressed significantly by treatment with either pinitol or E2. Femur weight, cellular bone rate, Ca and P content were improved by pinitol or E2. The DCI content of the serum of OVX decreased significantly, although it recovered to some extent after pinitol treatment. Pinitol significantly increased the ratio of DCI to MI in serum or bone marrow protein in the observed OVX mice. Besides, pinitol had no significant effects on osteoblast viability and differentiation. The present results showed that continuous pinitol intake exerts potent anti-osteoporosis activity via elevating DCI content in serum and bone marrow in OVX mice.

Estradiol Regulates Circadian Responses to Acute and Constant Light Exposure in Female Mice.

Sex hormones are well known to modulate circadian timekeeping as well as the behavioral and physiological responses to circadian disruption. Gonadectomy, reducing the amount of circulating gonadal hormones, in males and females produces alterations to the free-running rhythm and the responses to light exposure by the central oscillator of the suprachiasmatic nucleus (SCN). In this study, we tested whether estradiol plays a role in regulating the circadian responses to acute (light pulses) and chronic light exposure (constant light [LL] vs standard light:dark [LD] cycle) in female C57BL6/NJ mice. Mice were either ovariectomized or given sham surgery and given a placebo (P) or estradiol (E) pellet for hormone replacement so that there were 6 groups: (1) LD/Sham, (2) LL/Sham, (3) LD/OVX + P, (4) LL/OVX + P, (5) LD/OVX + E, and (6) LL/OVX + E. After 65 days of light cycle exposure, blood and SCNs were removed and serum estradiol plus SCN estradiol receptor alpha (ERα) and estradiol receptor beta (ERß) were measured via ELISA. The OVX + P mice exhibited shorter circadian periods and were more likely to become arrhythmic in LL compared with mice with intact estradiol (sham or E replacement mice). The OVX + P mice exhibited reduced circadian robustness (power) and reduced circadian locomotor activity in both LD and LL compared with sham controls or OVX + E mice. The OVX + P mice also exhibited later activity onsets in LD and attenuated phase delays, but not advances, when given a 15-min light pulse compared with estradiol intact mice. LL led to reductions in ERß, but not ERα, regardless of the surgery type. These results indicate that estradiol can modulate the effects of light on the circadian timing system and that estradiol can enhance responses to light exposure and provide protection against a loss of circadian robustness.

Loss of N-acetylglucosaminyl transferase V is involved in the impaired osteogenic differentiation of bone marrow mesenchymal stem cells.

The imbalance of bone resorption and bone formation causes osteoporosis (OP), a common skeletal disorder. Decreased osteogenic activity was found in the bone marrow cultures from N-acetylglucosaminyl transferase V (MGAT5)-deficient mice. We hypothesized that MGAT5 was associated with osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and involved in the pathological mechanisms of osteoporosis. To test this hypothesis, the mRNA and protein expression levels of MGAT5 were determined in bone tissues of ovariectomized (OVX) mice, a well-established OP model, and the role of MGAT5 in osteogenic activity was investigated in murine BMSCs. As expected, being accompanied by the loss of bone mass density and osteogenic markers (runt-related transcription factor 2, osteocalcin and osterix), a reduced expression of MGAT5 in vertebrae and femur tissues were found in OP mice. In vitro, knockdown of Mgat5 inhibited the osteogenic differentiation potential of BMSCs, as evidenced by the decreased expressions of osteogenic markers and less alkaline phosphatase and alizarin red S staining. Mechanically, knockdown of Mgat5 suppressed the nuclear translocation of ß-catenin, thereby downregulating the expressions of downstream genes c-myc and axis inhibition protein 2, which were also associated with osteogenic differentiation. In addition, Mgat5 knockdown inhibited bone morphogenetic protein (BMP)/transforming growth factor (TGF)-ß signaling pathway. In conclusion, MGAT5 may modulate the osteogenic differentiation of BMSCs via the ß-catenin, BMP type 2 (BMP2) and TGF-ß signals and involved in the process of OP.

Identification of kukoamine a as an anti-osteoporosis drug target using network pharmacology and experiment verification.

BACKGROUND: Osteoporosis (OP) is a major and growing public health problem characterized by decreased bone mineral density and destroyed bone microarchitecture. Previous studies found that Lycium Chinense Mill (LC) has a potent role in inhibiting bone loss. Kukoamine A (KuA), a bioactive compound extract from LC was responsible for the anti-osteoporosis effect. This study aimed to investigate the anti-osteoporosis effect of KuA isolated from LC in treating OP and its potential molecular mechanism. METHOD: In this study, network pharmacology and molecular docking were investigated firstly to find the active ingredients of LC such as KuA, and the target genes of OP by the TCMSP platform. The LC-OP-potential Target gene network was constructed by the STRING database and network maps were built by Cytoscape software. And then, the anti-osteoporotic effect of KuA in OVX-induced osteoporosis mice and MC3T3-E1 cell lines were investigated and the potential molecular mechanism including inflammation level, cell apoptosis, and oxidative stress was analyzed by dual-energy X-ray absorptiometry (DXA), micro-CT, ELISA, RT-PCR, and Western Blotting. RESULT: A total of 22 active compounds were screened, and we found KuA was identified as the highest active ingredient. Glycogen Phosphorylase (PYGM) was the target gene associated with a maximum number of active ingredients of LC and regulated KuA. In vivo, KuA treatment significantly increased the bone mineral density and improve bone microarchitecture for example increased BV/TV, Tb.N and Tb.Th but reduced Tb.Sp in tibia and lumber 4. Furthermore, KuA increased mRNA expression of osteoblastic differentiation-related genes in OVX mice and protects against OVX-induced cell apoptosis, oxidative stress level and inflammation level. In vitro, KuA significantly improves osteogenic differentiation and mineralization in cells experiment. In addition, KuA also attenuated inflammation levels, cell apoptosis, and oxidative stress level. CONCLUSION: The results suggest that KuA could protect against the development of OP in osteoblast cells and ovariectomized OP model mice and these found to provide a better understanding of the pharmacological activities of KuA again bone loss.

Fasting produces antidepressant-like effects via activating mammalian target of rapamycin complex 1 signaling pathway in ovariectomized mice.

Recent studies have shown that a 9-hour fast in mice reduces the amount of time spent immobile in the forced swimming test. However, whether 9-hour fasting has therapeutic effects in female mice with depressive symptoms has not been established. Therefore, in this study, we simulated perimenopausal depression via an ovariectomy in mice, and subjected them to a single 9-hour fasting 7 days later. We found that the ovariectomy increased the time spent immobile in the forced swimming test, inhibited expression of the mammalian target of rapamycin complex 1 signaling pathway in the hippocampus and prefrontal cortex, and decreased the density of dendritic spines in the hippocampus. The 9-hour acute fasting alleviated the above-mentioned phenomena. Furthermore, all of the antidepressant-like effects of 9-hour fasting were reversed by an inhibitor of the mammalian target of rapamycin complex 1. Electrophysiology data showed a remarkable increase in long-term potentiation in the hippocampal CA1 of the ovariectomized mice subjected to fasting compared with the findings in the ovariectomized mice not subjected to fasting. These findings show that the antidepressant-like effects of 9-hour fasting may be related to the activation of the mammalian target of the rapamycin complex 1 signaling pathway and synaptic plasticity in the mammalian hippocampus. Thus, fasting may be a potential treatment for depression.

Inhibitory Effects of Loganin on Adipogenesis In Vitro and In Vivo.

Obesity is characterized by the excessive accumulation of mature adipocytes that store surplus energy in the form of lipids. In this study, we investigated the inhibitory effects of loganin on adipogenesis in mouse preadipocyte 3T3-L1 cells and primary cultured adipose-derived stem cells (ADSCs) in vitro and in mice with ovariectomy (OVX)- and high-fat diet (HFD)-induced obesity in vivo. For an in vitro study, loganin was co-incubated during adipogenesis in both 3T3-L1 cells and ADSCs, lipid droplets were evaluated by oil red O staining, and adipogenesis-related factors were assessed by qRT-PCR. For in vivo studies, mouse models of OVX- and HFD-induced obesity were orally administered with loganin, body weight was measured, and hepatic steatosis and development of excessive fat were evaluated by histological analysis. Loganin treatment reduced adipocyte differentiation by accumulating lipid droplets through the downregulation of adipogenesis-related factors, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), perilipin 2 (Plin2), fatty acid synthase (Fasn), and sterol regulatory element binding transcription protein 1 (Srebp1). Loganin administration prevented weight gain in mouse models of obesity induced by OVX and HFD. Further, loganin inhibited metabolic abnormalities, such as hepatic steatosis and adipocyte enlargement, and increased the serum levels of leptin and insulin in both OVX- and HFD-induced obesity models. These results suggest that loganin is a potential candidate for preventing and treating obesity.

Glycyrrhizae radix et Rhizoma-Derived Carbon Dots and Their Effect on Menopause Syndrome in Ovariectomized Mice.

With the extension of the human life span and the increasing pressure of women's work and life, menopause syndrome (MPS) refers to a problem that puzzles almost all women worldwide. Hormone replacement treatment (HRT) can effectively mitigate the symptoms but can also exert adverse effects to a certain extent. Glycyrrhizae radix et rhizome (GRR) is commonly made into a charcoal processed product, termed GRR Carbonisatas (GRRC), for use in traditional Chinese medicine (TCM). GRRC is widely used to treat MPS and other gynecological diseases. In this study, GRRC was prepared through pyrolysis. Subsequently, GRR-derived carbon dots (GRR-CDs) were purified through dialysis and characterized using transmission electron microscopy, high-resolution transmission electron microscopy, Fourier-transform infrared, ultraviolet, fluorescence, X-ray photoelectron microscopy, and high-performance liquid chromatography. The effects of GRR-CDs on MPS were examined and confirmed using ovariectomized female mice models. The GRR-CDs ranged from 1.0 to 3.0 nm in diameter and with multiple surface chemical groups, as indicated by the results. GRR-CDs can elevate the estradiol (E2) level of healthy female mice. Moreover, GRR-CDs can alleviate MPS using the typical ovariectomized mice model, as confirmed by elevating the estradiol (E2) level and reducing the degree of follicle stimulating hormone (FSH) and luteinizing hormone (LH) and raising the degree of uterine atrophy. The results of this study suggested that GRR-CDs may be a potential clinical candidate for the treatment of MPS, which also provides a possibility for nanodrugs to treat hormonal diseases.

Cordyceps sinensis extract protects against the ovariectomy-induced bone loss via the action on osteoclasts / 药学实践杂志

Objective To explore the effects of Cordyceps sinensis extract (CSE) on osteoporosis and RANKL-mediated osteoclastogenesis. Methods Bone marrow-derived macrophages (BMMs) was isolated from the bone marrow of C57BL/6 mice. CSE was added in osteoclast differentiation. Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP). The nearly mature osteoclasts were planted on hydroxyapatite plates and the area of bone lacunae was observed by microscope. The F-actin belt was stained by DAPI and phylloeptide and the number of nuclei was observed by confocal microscopy. The expressions of DC-STAMP, ATP6V0D2, TRAP, CTSK, and NFATC1 were detected by q-PCR. The protein expression of the MAPK pathway was detected by Western Blot. The in vivo experiments were carried out by administering CSE to the ovariectomized mice daily through gavage. After 6 weeks of intervention, mouse femurs were taken for morphological analysis. Peripheral blood was taken for ELISA. Results CSE represses osteoclastogenesis, bone resorption, F-actin belts formation, osteoclast specific gene expressions and MAPK signaling pathways in vitro. In vivo study indicated that CSE prevents OVX-induced osteoporosis and preserves bone volume by repressing osteoclast activity and function. It also increases the serum ALP, BGP content, and reduces TRAP content. Conclusion CSE can attenuate osteoclast formation and OVX-induced osteoporosis, suggesting potential clinical therapeutic effects for osteoporosis.

Integration of pharmacodynamics and metabolomics reveals the therapeutic effects of 6-acetylacteoside on ovariectomy-induced osteoporosis mice.

BACKGROUND: 6-acetylacteoside (6-AA) is a phenylethanoid glycoside isolated from Cistanche deserticola which had been previously proven to possess anti-osteoporotic activity previously. Currently, it is still unknown whether 6-AA plays a crucial role on the anti-osteoporotic effects of C. deserticola. PURPOSE: To elucidate the therapeutic effect and mechanism of 6-AA on osteoporosis by employing an ovariectomized mouse model in vivo and RAW264.7 cells in vitro. METHODS: Sixty female ICR mice were randomly assigned into six groups: sham-operated control group (SHAM, vehicle), ovariectomized model group (OVX, vehicle), positive group (EV, 1 mg/kg/day of estradiol valerate), low dosage (10 mg/kg/day of 6-AA), medium dosage (20 mg/kg/day of 6-AA) and high dosage (40 mg/kg/day of 6-AA) treatment groups. All substances were administered daily by intragastric gavage. After 12 weeks of intervention, trabecular bone microarchitecture was estimated and bone biomechanics were determined. Bone formation and resorption factors were determined by using the corresponding Elisa kits. The related proteins and metabolites were estimated by using western-blot and metabolomics techniques. RESULTS: OVX mice demonstrated significant atrophy of the uterine and vagina, declined biomechanical parameters such as flexural strength and maximum load, deteriorated trabecular bone microarchitecture such as decreased BMD, BMC, TMC, TMD, BVF, Tb. N, and Tb. Th and increased Tb. Sp, as well as increased bone resorption factors such as TRAP, cathepsin K, and DPD, all after 12 weeks of ovariectomy operation. Following administration of 6-AA to OVX mice, parameters related to the bone microarchitecture, bone resorption activities as well as biomechanical properties were all significantly improved. Meanwhile, the levels of NF-κB, NFATc1, RANK, RANKL and TRAF6 were significantly downregulated, while OPG, PI3K and AKT were upregulated after 6-AA intervention. This indicates that, 6-AA could prevent bone resorption by regulating the RANKL/RANK/OPG mediated NF-κB and PI3K/AKT pathways. Furthermore, 26 different metabolites corresponding to 25 metabolic pathways were identified, and 5 of which were related to the formation of osteoporosis. Interestingly, 23 abnormal metabolites were recovered after 6-AA treatment. CONCLUSION: Our results revealed the significant anti-osteoporotic effects of 6-AA on ovariectomized mice which were probably exerted via suppression of osteoclast formation and bone resorption.

Anti-Osteoporosis Effects of the Fruit of Sea Buckthorn (Hippophae rhamnoides) through Promotion of Osteogenic Differentiation in Ovariectomized Mice.

The fruit of Hippophae rhamnoides has been widely used for medicinal purposes because of its anti-inflammatory, antioxidant, antiplatelet, and antimicrobial effects. Since there are no clear reports on the therapeutic efficacy of H. rhamnoides in osteoporosis, this study aimed to confirm the potential use of H. rhamnoides for the treatment of osteoporosis through its osteogenic differentiation-promoting effect in ovariectomized mice. Through an in vitro study, we compared the effects of the EtOH extract of H. rhamnoides fruits (EHRF) on the differentiation of C3H10T1/2, a mouse mesenchymal stem cell line, into osteoblasts based on alkaline phosphatase (ALP) staining and the relative expression of osteogenesis-related mRNAs. The EHRF significantly stimulated the differentiation of mesenchymal stem cells into osteoblasts and showed 7.5 times (* p < 0.05) higher osteogenesis than in the untreated control. A solvent fractionation process of EHRF showed that the hexane-soluble fraction (HRH) showed 10.4 times (** p < 0.01) higher osteogenesis than in the untreated control. Among the subfractions derived from the active HRH by preparative HPLC fractionation, HRHF4 showed 7.5 times (* p < 0.05) higher osteogenesis than in the untreated naïve cells, and HRH and HRHF4 fractions showed 22.6 times (*** p < 0.001) stronger osteogenesis activity than in the negative control. Osteoporosis was induced by excision of both ovaries in 9-week-old female ICR mice for in vivo analysis, and two active fractions, HRH and HRHF4, were administered orally for three months. During the oral administration period, body weight was measured weekly, and bone mineral density (BMD) and body fat density were measured simultaneously using a DEXA machine once a month. In particular, during the in vivo study, the average BMD of the ovariectomized group decreased by 0.0009 g/cm2, whereas the average BMD of the HRH intake group increased by 0.0033 g/cm2 (* p < 0.05) and that of the HRHF4 intake group increased by 0.0059 g/cm2 (** p < 0.01). The HRH and HRHF4 intake groups significantly recovered the mRNA and protein expression of osteogenic genes, including ALP, Osteopontin, Runx2, and Osterix, in the osteoporosis mouse tibia. These findings suggest that the active fractions of H. rhamnoides fruit significantly promoted osteoblast differentiation in mesenchymal stem cells and increased osteogenic gene expression, resulting in an improvement in bone mineral density in the osteoporosis mouse model. Taken together, H. rhamnoides fruits are promising candidates for the prevention and treatment of osteoporosis.

Di-(2-ethylhexyl) phthalate exposure links to inflammation and low bone mass in premenopausal and postmenopausal females: Evidence from ovariectomized mice and humans.

OBJECTIVE: Phthalates induce inflammation and are ubiquitously used in daily life. We aim to study the impact of di-(2-ethylhexyl) phthalate (DEHP) exposure on inflammation and osteoporosis in premenopausal and postmenopausal females. METHODS: Female 8-week-old C57BL/6JNarl mice received an ovariectomy (OVX) or a sham operation and were fed with DEHP or vehicle by oral gavage for 4 or 8 weeks. Their femurs were isolated for micro-computed tomography, and their serum was collected for inflammatory cytokine assays. Correlations between urinary phthalate metabolites and the lumbar spine bone mineral density (BMD) in premenopausal and postmenopausal volunteers were performed. RESULTS: Among the OVX mice treated for 4 weeks, significant lower bone volume, bone volume/tissue volume, and trabecular number but significant higher trabecular bone pattern factor and structure model index were identified in the mice treated with DEHP than with vehicle. The OVX mice treated with DEHP for 4 weeks had significantly higher serum interleukin (IL)-1ß, IL-10, IL-17A, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and Dickkopf-1 levels than those treated with vehicle. The sham mice treated with DEHP for 8 weeks showed an impaired femur trabecular microstructure and had significantly higher serum IL-1ß, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α than those treated with vehicle. DEHP metabolites were inversely correlated with the BMD of premenopausal women and the T-score of postmenopausal women. CONCLUSION: DEHP treatment in OVX and sham mice results in osteoporosis and impairs the microstructure of the femur trabecula through inflammation. Phthalate exposure negatively affects the bone mass in both premenopausal and postmenopausal women. Thus, long-term avoidance is suggested.

(S)-Equol Is More Effective than (R)-Equol in Inhibiting Osteoclast Formation and Enhancing Osteoclast Apoptosis, and Reduces Estrogen Deficiency-Induced Bone Loss in Mice.

BACKGROUND: Equol, a metabolite of daidzein, binds to the estrogen receptor with greater affinity than daidzein and exhibits various biological properties. It exists as an enantiomer, either (S)-equol or (R)-equol. OBJECTIVES: We have previously shown that the inhibitory effect of (S)-equol on bone fragility is stronger than that of racemic equol in ovariectomized (OVX) mice; however, the effect of (R)-equol has not been elucidated. The aim of this study was to compare the activities of equol enantiomers on bone metabolism in vitro and in vivo. METHODS: Bone marrow cells (BMCs) and RAW 264.7 cells were treated with equol enantiomers. The number of osteoclasts and caspase-3/7 activity were measured. We examined the effect of equol enantiomers on osteoblast differentiation in MC3T3-E1 cells. In vivo, 8-wk-old female ddY mice were assigned to 4 groups: sham-operated (sham), OVX, OVX + 0.5 mg/d of (S)-equol (S-eq), and OVX + 0.5 mg/d of (R)-equol (R-eq). Four weeks after the intervention, femoral bone mineral density (BMD) and osteoclastic gene expression were analyzed, along with concentrations of equol enantiomers in the serum and tissues. RESULTS: (S)-equol and (R)-equol inhibited osteoclast differentiation in BMCs (97% and 60%, P < 0.05) and RAW 264.7 cells (83% and 68%, P < 0.05). (S)-equol promoted apoptosis of mature osteoclasts by inducing caspase-3/7 activity (29%, P < 0.05) and enhanced osteoblast differentiation (29%, P < 0.05). In OVX mice, BMD was ameliorated in (S)-equol-treated mice (11%, P < 0.05), but not in (R)-equol-treated mice. The concentrations of (S)-equol were greater than those of (R)-equol in the serum, tibia, liver, and kidney (by 148%, 80%, 22%, and 139%, respectively). CONCLUSIONS: These results suggest that (S)-equol is more effective than (R)-equol in inhibiting osteoclast formation and enhancing osteoclast apoptosis in vitro, supporting the beneficial effect of (S)-equol to reduce estrogen deficiency-induced bone loss in OVX mice.

Neuronal GPER Participates in Genistein-Mediated Neuroprotection in Ischemic Stroke by Inhibiting NLRP3 Inflammasome Activation in Ovariectomized Female Mice.

Estrogen replacement therapy (ERT) is potentially beneficial for the prevention and treatment of postmenopausal cerebral ischemia but inevitably increases the risk of cerebral hemorrhage and breast cancer when used for a long period of time. Genistein, a natural phytoestrogen, has been reported to contribute to the recovery of postmenopausal ischemic stroke with reduced risks. However, the underlying mechanism of genistein-mediated neuroprotection remains unclear. We reported that genistein exerted significant neuroprotective effects by enhancing the expression of neuronal G protein-coupled estrogen receptor (GPER) in the ischemic penumbra after cerebral reperfusion in ovariectomized (OVX) mice, and this effect was achieved through GPER-mediated inhibition of nod-like receptor protein 3 (NLRP3) inflammasome activation. In addition, we found that peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) was the pivotal molecule that participated in GPER-mediated inhibition of NLRP3 inflammasome activation in OVX mice after ischemia/reperfusion (I/R) injury. Our data suggest that the neuronal GPER/PGC-1α pathway plays an important role in genistein-mediated neuroprotection against I/R injury in OVX mice.

Inhibitory Effect of Ulmus davidiana and Cornus officinalis Extracts on Osteoporotic Bone Loss In Vitro and In Vivo.

Background and Objectives: Traditional herbal medicines are becoming more popular as a complementary medication as they have the advantages of being mostly harmless and safe, causing fewer side-effects than conventional medications. Here, we demonstrate the inhibitory effects of the combination of Ulmus davidiana (UD) and Cornus officinalis (CO) extracts on osteoporotic bone loss. Materials and Methods: This study presented osteogenic effects in primary cultured osteoblasts, pre-osteoblastic MC3T3-E1 cell lines, and osteoclastogenic effects in osteoclasts derived from bone marrow monocytes, and finally, protective effects on bone loss in an ovariectomy (OVX)-induced osteoporotic animal model. Results: A significant increase in alkaline phosphatase (ALP) activity was observed following treatment with UD and CO mixtures (8:2, 7:3, and 5:5 ratios) and individual UD and CO extracts, with the highest ALP activity being detected for the treatment with UD and CO extracts at a 5:5 ratio. An optimal ratio of UD and CO (UC) extract promoted osteoblast differentiation in both pre-osteoblastic cells and primary osteoblasts by increasing osteoblastic markers such as Alpl, Runx2, and Bglap. However, treatment with the UC extract inhibited osteoclast differentiation with a decreased expression of osteoclastogenesis-related genes, including Ctsk, Acp5, Mmp9, and Nfatc1. In addition, UC treatment prevented osteoporotic bone loss in OVX mice and improved impaired skeletal structure parameters. Conclusions: This study suggests that combined UD and CO extracts may be a beneficial traditional medicine for the prevention of postmenopausal osteoporosis.

Estrogen exposure causes the progressive growth of SK-Hep1-derived tumor in ovariectomized mice.

Liver cancer, one of the leading death causes, has different incidence and mortality rates in men and women. The influencing factor is considered to estrogen. However, the role of estrogen in liver cancer remains controversial. In this study, we investigated the effects of estrogen on tumor progression. Total RNA sequencing was analyzed in SK-Hep1-derived tumor tissues, and 15 genes were expressed only in female mice. Among the differentially expressed genes, matrix metalloprotease 7 (MMP7), germ cell associated 1 (GSG1), and chromosome 6 open reading frame 15 (C6orf15) were associated with significantly different overall survival rates based on their expression level in liver cancer patients. Interestingly, exogenous estrogen aggravated SK-Hep1-derived tumor growth in ovariectomized (OVX) mice. When OVX mice were treated with exogenous estrogen, SK-Hep1-derived tumor tissues exhibited high MMP7 expression levels and low GSG1 and C6orf15 expression levels. These expression patterns were consistent with those of liver cancer patients with low overall survival rates. These results suggest that these genes are expected to be prognostic biomarkers of liver cancer. In conclusion, our results suggest that continuous estrogen exposure may promote tumor growth in OVX mice.

Effect of Medicago sativa compared To 17ß-oestradiol on osteoporosis in ovariectomized mice.

Phytoestrogens, with a wide range of beneficial effects, prevent bone loss caused by oestrogen deficiency.The purpose of this study was to evaluate the effect of Medicago sativa ethanol extract compared to 17ß-oestradiol on osteoporosis in ovariectomized mice.The study was carried out on female mice, divided into five groups: control mice (GI), Medicago sativa treated mice (0.75 g/kg BW/day) (GII), ovariectomized mice (GIII) and ovariectomized mice treated either with Medicago sativa (GIV) or with 17ß-oestradiol (50 µg/Kg BW/day) (GV).Our results showed that Medicago sativa or 17ß-oestradiol treatments significantly attenuated perturbations of mineral levels, histological changes and oxidative stress in the femurs of ovariectomized mice.Medicago sativa prevented bone loss induced by oestrogen deficiency, which could be attributed to its richness in kaempferol, syringic acid, naringenin and myrictin. Its effects were more beneficial or similar compared to 17ß-oestradiol.